Constructs for continuous monitoring of live cells

ABSTRACT

The present invention provides for methods to obtain multiple information-rich samples at different time points from the same cell while minimally disrupting the cell. The subject matter disclosed herein is generally related to nucleic acid constructs for continuous monitoring of live cells. Specifically, the subject matter disclosed herein is directed to nucleic acid constructs that encode a fusion protein and a construct RNA sequence that induce live cells to self-report cellular contents while maintaining cell viability. The present invention may be used to monitor gene expression in single cells while maintaining cell viability.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/397,867 filed on Sep. 21, 2016. The entire contents of theabove-identified application are fully incorporated herein by reference.

TECHNICAL FIELD

The subject matter disclosed herein is generally related to nucleic acidconstructs for continuous monitoring of live cells. Specifically, thesubject matter disclosed herein is directed to nucleic acid constructsthat encode a fusion protein and a construct RNA sequence that inducelive cells to self-report cellular contents while maintaining cellviability.

BACKGROUND

Single-cell gene expression (SCGE) profiling is an important analyticaltechnique for the study of mammalian cells. The ability to obtain highlyresolved molecular phenotypes directly from individual cells istransforming the way cell states are defined, cell circuitry isunderstood, and how cellular responses to environmental cues arestudied. There is tremendous interest in moving beyond static snapshotsof SCGE in cell suspensions to understand how SCGE profiles change overtime. Technology that reports the internal state and functional historyof cells within tissues would enable novel insight into dynamicbiological processes. Current SCGE profiling technology addresses staticheterogeneity (e.g. a snapshot of differences among single cells).However, dynamic signaling processes (Cai L, Nature 2008; Yosef N, Cell2011; Yosef N, Nature 2013) and transitions in cell type and functionover time are crucial to cellular biology and organism-level function.Enabling the comprehensive study of dynamic processes at the single-celllevel is of intense interest, but tools for non-destructive in situanalysis are currently lacking. New methods are needed to obtainmultiple information-rich samples at different time points from the samecell while minimally disrupting the cell.

SUMMARY

In one aspect, the embodiments described herein are directed to nucleicacid constructs that encode a fusion protein and a construct RNAsequence. The fusion protein may comprise a secretion-inducing domainand a construct RNA capture domain. When expressed in live cells thesecretion domain induces the cell to export samples of cellular contentthat can be isolated and analyzed while maintaining cell viability. Incertain example embodiments, the secretion domain facilitates theformation of an export compartment capable of packaging cellularcontents and exporting those cellular contents from the cell. Theconstruct RNA capture domain of the fusion protein is one member of abinding pair that binds a corresponding RNA retrieval element on theexpressed construct RNA sequence. The construct RNA sequence comprises aconstruct RNA retrieval element and a cellular RNA capture element. Theconstruct RNA sequence may further comprise a barcode. The construct RNAretrieval element is recognized and bound by the construct RNA capturedomain of the fusion protein. The cellular RNA capture domain hybridizesto cellular RNA. Binding of the construct RNA sequence/cellular RNAcomplex by the construct RNA capture element of the fusion proteinresults in export of the construct RNA sequence/cellular RNA complex inassociation with the secretion-inducing domain of the fusion protein.Thus, capture of cellular RNA by the construct RNA sequence enablesexport of the captured cellular RNA in association with thesecretion-inducing domain of the fusion protein. In certain exampleembodiments, the secretion-inducing domain is a viral capsid or coatprotein. In certain example embodiments, the secretion-inducing domaincomprises a Gag protein or a functional fragment thereof. In certainexample embodiments, the construct RNA capture domain of the fusionprotein is a MS2 coat protein and the construct RNA retrieval element ofthe construct RNA comprises a sequence encoding a MS2 hairpin. Incertain example embodiments, the construct RNA capture domain of thefusion protein is dCas9 and the construct RNA retrieval element of theconstruct RNA is a dCas9 binding loop.

In certain example embodiments, the RNA construct may further comprise abarcode, and a poly U sequence or a sequence comprising a (UUG)n motiffor capture of cellular RNA. The barcode comprises a randomized sequenceunique to the construct and therefore to the cell or cell population theconstruct is delivered to. Thus, in certain example embodiments, allcellular RNA captured by the RNA construct and exported from the cellvia the fusion protein will have the same barcode thereby identifyingall cellular RNA exported from the same cell.

The nucleic acid constructs described herein may further comprise aninducible promoter to control expression of the fusion protein, and/orconstruct RNA sequence. In certain example embodiments, the promoter maybe a tissue or cell-specific promoter. The nucleic acid constructsdescribed herein may further comprise a steric linker. The steric linkermay be located on a N-terminus of the secretion-inducing protein orbetween the secretion-inducing domain and the construct RNA capturedomain and may control the rate of secretion, the size of exportcompartments formed by the secretion-inducing protein, or both. Thenucleic acid constructs described herein may further encode a fusionprotein that includes an affinity tag for subsequent isolation andenrichment of the fusion protein and/or export compartments formed bythe fusion protein. Further, the nucleic acids constructs may encode adetectable self-reporting molecule that can be used to confirmsuccessful delivery and expression of the nucleic acid constructsdescribed herein. In certain example embodiments, the detectableself-reporting molecule may be a cleavable self-reporting molecule thatcan be cleaved from the RNA construct after expression.

In another aspect, the embodiments disclosed herein comprise methods forcontinuous monitoring of live cells comprising delivering into a cell anucleic acid construct described herein. The nucleic acid construct isexpressed, for example, via an inducible promoter. Cellular RNA, such asmRNA or microRNA, is captured by hybridization to the cellular RNAcapture element of the construct RNA sequence. The captured cellular RNAis then exported from the cell by binding of the construct RNA capturedomain of the fusion protein to the retrieval element of the constructRNA sequence such that the construct RNA sequence—and bound cellularRNA—are exported from the cell in association with secretion inducingdomain of the cellular protein. The exported fusion protein/constructRNA sequence/cellular RNA complex may then be isolated.

In certain example embodiments, the method further comprises generatinga RNA-DNA duplex by reverse transcribing the captured cellular RNA usingthe construct RNA sequence as a primer for reverse transcription. ADNA-DNA duplex is then generated by converting the construct RNAsequence to a corresponding DNA sequence with second strand synthesisusing a DNA primer. The DNA-DNA duplex is then used to generate asequencing library for sequencing using, for example, a NGS sequencingplatform. Sequencing of the DNA-DNA duplex library identifies thetranscript and—via the barcode information—the cell of origin for eachtranscript thereby enabling continuous single cell gene expressionanalysis.

In certain example embodiments, a nucleic acid construct for barcodingcellular components, such as expressed RNAs, comprises a barcode and acellular RNA capture element. In certain example embodiments, thecellular RNA capture element is a poly(U) or (UUG)_(n) motif. In certainexample embodiments, the nucleic acid construct may further comprise afilter sequence that helps identify the barcode sequence in downstreamsequencing reads. In certain example embodiments, the nucleic acidconstruct may comprise an adapter sequence that provides a complementarybinding site for a reverse transcription or amplification primer. Incertain other example embodiments, the nucleic acid construct mayfurther comprise a sequencing primer binding site that is complementaryto one or more sequencing primers used in downstream sequencingreactions. The nucleic acid constructs described in this paragraph maybe used as the construct RNA sequence in relation to the self-reportingexport compartment embodiments discussed above.

In another aspect, a method for labeling molecular components of cellsaccording to cell or origin comprises expressing any of the abovedisclosed nucleic acid constructs in one or more cells, wherein theexpressed nucleic acid construct comprises a barcode that is unique toan individual cell or cell lineage, capturing cellular RNA expressed inthe one or more cells by binding of the cellular RNA via the cellularRNA capture element of the expressed construct sequence andincorporating the barcode of the expressed nucleic acid construct to thecaptured cellular RNA to generate barcoded cellular RNA. Barcoded RNArefer to directly barcoded RNAs as well as single and double strandedcopies made from the original cellular RNA such as those shown in FIGS.12-15. The barcode may be attached by ligation of the nucleic acidconstruct to the cellular RNA by RNA-RNA ligation, by priming firstand/or second strand synthesis of the captured cellular RNA using theexpressed nucleic acid construct. Barcoded RNA may be further amplified,for example, by RNA-dependent RNA synthesis, PCR, or linear DNAamplification.

In another aspect, the embodiments disclosed herein comprise vectorscomprising the nucleic acid constructs described herein. In certainexample embodiments, the vectors are viral vectors. In certain otherexample embodiments, the vectors are non-viral vectors.

In another aspect, embodiments disclosed herein include kits comprisingthe nucleic acid constructs and/or vectors described herein.

These and other aspects, objects, features, and advantages of theexample embodiments will become apparent to those having ordinary skillin the art upon consideration of the following detailed description ofillustrated example embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—is a schematic depicting a method for continuous single cell geneexpression analysis of live cells, in accordance with certain exampleembodiments.

FIG. 2—is a diagram depicting a barcoded self-reporting strategy inaccordance with certain example embodiments.

FIG. 3—is a diagram of a construct in accordance with certain exampleembodiments. The diagram shows a possible DNA construct for making Gagfusion proteins. The glycine-serine (GS) linker (SEQ ID NO: 7) functionsas a flexible amino acid linker between the gag protein and the clonedprotein of interest. The RNA capture domain of interest is ligated intothe construct in the multiple cloning site (MCS) via standardrestriction cloning techniques. The p2A linker (SEQ ID NO: 5) serves asa self-cleaving linker, allowing yellow fluorescent protein (YFP) (SEQID NO: 6) to be translated from the same transcript without fusion TheDNA construct includes a bGH pA terminator (SEQ ID NO: 8). The constructmay include a spacer between elements (SEQ ID NO: 9)

FIG. 4—is a schematic of single cell expression analysis using anexample inducible construct further encoding a construct self-reportingmolecule that may be used to indicate successful delivery to targetcells, in accordance with certain example embodiments.

FIG. 5—is a schematic showing an example construct comprising atissue-specific promoter, a dox-inducible promoter or a combination ofthe two, a linker, and labile self-reporting molecule and the use ofsaid construct in accordance with certain example embodiments.

FIG. 6—is a schematic of an example construct further encoding anaffinity tag for subsequent isolation and enrichment of expressed VLPsin accordance with certain example embodiments.

FIG. 7—is a diagram summarizing simulation of export compartment sizeand the theoretical number of mRNA that could be packaged inside anexample export compartment.

FIG. 8—is a graph showing a simulation based on exclusive reads per celltype that allows for >80% accuracy of prediction with a simple algorithmthat uses inner-products and training on 10 cells per cell type.

FIG. 9—is a graph showing the percent of the proteome that is composedof Gag proteins per number of transcripts sampled.

FIG. 10—is a table showing projected achievable time resolution of geneexpression using the constructs described herein.

FIG. 11—is a schematic showing one example embodiment for incorporationof barcodes of dsDNA amplicons derived from cellular mRNA isolated fromexport compartments.

FIG. 12—is a schematic showing one example embodiment for incorporationof barcodes into dsDNA amplicons derived from cellular mRNA isolatedfrom export compartments.

FIG. 13—is a schematic showing one example embodiment for incorporationof barcodes into dsDNA amplicons derived from cellular mRNA isolatedfrom export compartments.

FIG. 14—is a schematic showing one example embodiment for incorporationof barcodes into dsDNA amplicons derived from cellular mRNA isolatedfrom export compartments.

FIG. 15—A) Reverse transcription with RNA primers. B) Reversetranscription in crosstalk-preventing hydrogels with RNA primers. C)Genomic integration of synthetic RNA barcodes in HEK cells by lentiviraltransduction. D) Efficient in vitro library construction of RNA barcodedmonoclonal RNA template. The filter may include a Smart-seq2 handle (SEQID NO: 11).

FIG. 16—A) Gag-MCP (Gag-MS2) forms VLPs as demonstrated by an anti-Gagwestern supernatant. B) Pol III driven RNA barcodes transcripts containa 5′ rev response element and are co-expressed with Rev viral proteinsfor nuclear export. RNA barcode transcripts are engineered with MS2hairpins for binding to the MS2 coat protein (MCP) domain within gag-MCPfusion proteins. Barcodes are expressed within wild-type gag expressingcells (to serve as a measure of background export) and within gag-MCPexpressing cells for directed export within gag-MCP VLPs. Barcodeseither contain a 3′ poly(U) tail for hybridizing to polyadenylated RNAsor a scrambled 3′ tail as a hybridization control. C) Gag-MCP VLPssuccessfully package and export endogenous mRNA, as measured by GAPDHRT-qPCR.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS General Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Definitions of common termsand techniques in molecular biology may be found in Molecular Cloning: ALaboratory Manual, 2^(nd) edition (1989) (Sambrook, Fritsch, andManiatis); Molecular Cloning: A Laboratory Manual, 4^(th) edition (2012)(Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (AcademicPress, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B.D. Hames, and G. R. Taylor eds.): Antibodies, A Laboraotry Manual (1988)(Harlow and Lane, eds.): Antibodies A Laboraotry Manual, 2^(nd) edition2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney,ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008(ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829);Robert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 9780471185710); Singleton et al., Dictionary of Microbiology andMolecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed.,John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Janvan Deursen, Transgenic Mouse Methods and Protocols, 2^(nd) edition(2011).

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The term “optional” or “optionally” means that the subsequent describedevent, circumstance or substituent may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances where it does not.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The terms “about” or “approximately” as used herein when referring to ameasurable value such as a parameter, an amount, a temporal duration,and the like, are meant to encompass variations of and from thespecified value, such as variations of +/−10% or less, +/−5% or less,+/−1% or less, and +/−0.1% or less of and from the specified value,insofar such variations are appropriate to perform in the disclosedinvention. It is to be understood that the value to which the modifier“about” or “approximately” refers is itself also specifically, andpreferably, disclosed.

Reference throughout this specification to “one embodiment”, “anembodiment,” “an example embodiment,” means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention. Thus,appearances of the phrases “in one embodiment,” “in an embodiment,” or“an example embodiment” in various places throughout this specificationare not necessarily all referring to the same embodiment, but may.Furthermore, the particular features, structures or characteristics maybe combined in any suitable manner, as would be apparent to a personskilled in the art from this disclosure, in one or more embodiments.Furthermore, while some embodiments described herein include some butnot other features included in other embodiments, combinations offeatures of different embodiments are meant to be within the scope ofthe invention. For example, in the appended claims, any of the claimedembodiments can be used in any combination.

All publications, published patent documents, and patent applicationscited herein are hereby incorporated by reference to the same extent asthough each individual publication, published patent document, or patentapplication was specifically and individually indicated as beingincorporated by reference.

Overview

Embodiments disclosed herein provide nucleic acid constructs and methodsof use thereof that induce a live cell to self-report sub-samples ofcellular content. The sampling can be general or can be targeted to aparticular class of molecules or to specific types of molecules. Theconstructs facilitate generation of a read-out for high-throughputscreens by combining engineered export with simple bulk sample andsample processing. Live cell sampling enables time course measurementsand expands, for example, the applicability of transcriptional profilesobtained by single cell gene expression analysis. The constructs mayfurther comprise steric linkers, inducible promoters, detectableself-reporting molecules, and affinity elements as discussed in furtherdetail below. When introduced into live cells the constructs disclosedherein enable live cell sampling of cellular contents while maintainingcell viability. Cellular contents may include nuclear as well ascytosolic contents. In addition, the nucleic acid constructs and methodsfurther comprise the use of nucleic acid barcodes that tag eachtranscript molecule with a cell-identifying barcode, adding single-celltranscriptomic analysis to the self reporting approach disclosed herein.

Nucleic Acid Constructs

In certain example embodiments, the nucleic acid constructs comprise anucleic acid sequence encoding a fusion protein and a construct RNAsequence. The fusion protein comprises a secretion-inducing domain and aconstruct RNA capture domain.

A secretion-inducing protein may comprise a polypeptide that whenexpressed induces a cell to export cellular contents in association withthe secretion-inducing protein. As used herein, and in the context ofproteins encoded by the nucleic acid constructs described herein, a“protein” may refer to the full length sequence of the protein or onlythat portion of the protein that is necessary for the function for whichthe full length protein is otherwise expressed. In certain exampleembodiments, the secretion-inducing protein is an export compartmentprotein. An export compartment protein may be any protein thatself-assembles upon expression in a cell into an export compartment. Incertain example embodiments, an export compartment is a sphericalmacromolecular assembly comprising a protein inner layer and an outerlipid containing membrane, with at least the export-compartment proteinforming the inner protein layer. In certain example embodiments, theexport compartment protein may only form a partial export compartmentwhile retaining the ability to associate with and export the targetedcellular contents. In certain example embodiments, the exportcompartment protein is a viral export compartment protein that formsvirus-like particles. Regarding embodiments that use viral exportcompartment proteins, the terms export compartment and virus-likeparticle (VLP) may be used interchangeably. Example viral exportcompartment proteins may include viral capsid proteins. In certainexample embodiments, the viral capsid protein is a viral Gag protein. Incertain example embodiments, the viral Gag protein is a lentivirus Gagprotein. In certain example embodiments, the export compartment proteinis encoded by a nucleic acid sequence of SEQ ID NO: 1.

The construct RNA capture domain may be a protein or peptide thatrecognizes and binds a retrieval element of the construct RNA sequenceafter expression of the construct RNA sequence in the cell. Theconstruct RNA capture domain of the fusion protein may comprise anyprotein or peptide that recognizes and selectively binds a targetsequence or structural feature of the expressed construct RNA sequence.In certain example embodiments, the construct RNA capture domain may bea protein or peptide that recognizes and binds RNA secondary structuralfeatures, such as but not limited to, hairpins. In certain exampleembodiments, the construct RNA capture domain comprises a dCas9 proteinand the retrieval element of the construct RNA sequence may comprise asequence encoding the dCas9-binding hairpin. In certain other exampleembodiments, the construct RNA capture domain of the fusion protein maybe a viral capsid protein that binds a sequence or structural feature ofthe corresponding viral genome. For example, the construct RNA capturedomain may be a MS2 coat protein and the retrieval element of theconstruct RNA sequence may comprise a RNA sequence defining a MS2hairpin. In certain example embodiments, the construct RNA capturedomain comprises a protein encoded by SEQ ID NO: 2, SEQ ID NO: 3, or SEQID NO: 4, or functional equivalents thereof. In certain exampleembodiments, the retrieval element of the construct RNA sequencecomprises SEQ ID NO: 10.

The construct RNA sequence comprises a retrieval element and a cellularRNA capture element. The construct RNA may also further comprise areverse transcription primer binding site and a barcode. The constructRNA retrieval element is recognized and bound by the construct RNAcapture domain on the fusion protein such that the construct RNA isexported from the cell in association with the secretion-inducingprotein. In certain example embodiments, the secretion-inducing proteinis an export compartment protein and the construct RNA is packagedwithin the export compartment formed by the fusion protein. In certainexample embodiments, the cellular RNA capture element hybridizes tocellular RNA such that the bound cellular RNA is packaged inside theexport compartment with the construct RNA.

The cellular RNA capture element of the construct RNA sequence bindstarget RNAs in the cell. The cellular RNA capture element may bindtarget RNAs in an unbiased manner. For example, the cellular RNA captureelement may be a poly-U sequence. In certain example embodiments, thepoly-U sequence is approximately 15 to approximately 50 nucleotideslong. In certain other example embodiments, the cellular RNA captureelement may comprise a (UUG)n motif, wherein “n” may range fromapproximately 1 to approximately 20. In certain example embodiments, thecellular RNA capture element may comprise a sequence that can hybridizeto a specific target RNA species, such as specific mRNA transcript. Incertain example embodiments, the cellular RNA capture element comprisesSEQ ID NO: 12.

The construct RNA sequence may further include a barcode. A barcode isgenerated by sequentially attaching two or more detectableoligonucleotide tags to each other. As used herein, a “detectableoligonucleotide tag” is an oligonucleotide that can be detected bysequencing of its nucleotide sequence and/or by hybridization todetectable moieties such as optically labeled probes. Theoligonucleotide tags that make up a barcode are typically randomlyselected from a diverse set of oligonucleotide tags. For example, anoligonucleotide tag may be selected from a set A, B, C, and D, with eachset comprising random sequences of a particular size. An oligonucleotidetag is first selected from set A, then a second oligonucleotide tag isselected from set B and concatenated to the oligonucleotide from set A.The process is repeated for sets C and D such that an oligonucleotidetag from C is concatenated to AB and an oligonucleotide tag from D isconcatenated to ABC. The particular sequence selected from each set andthe order in which the oligonucleotides are concatenated define a uniquebarcode. Methods for generating barcodes for use in the constructsdisclosed herein are described, for example, in International PatentApplication Publication No. WO/2014/047561. In certain exampleembodiments, the barcodes are approximately 10 to approximately 40nucleotides long. In certain example embodiments, the barcodes comprise2, 3, 4, 5, 6, 7, 8, 9, or 10 distinct ordered positions. In certainexample embodiments, the barcode of each construct is unique to thatconstruct or sub-set of constructs such that delivery of that constructor sub-set of constructs is unique to that cell or population of cells.For example, a first cell or population of cells may be transduced witha first construct or set of constructs comprising a first barcode, and asecond cell or second population of cells may be transduced with asecond construct of set of constructs comprising a second barcode, suchthat sequencing libraries derived from exported cellular RNA from aparticular cell or cell population will include the same unique barcode,thereby identifying those cellular RNAs as originating from the samecell or same cell population.

In certain example embodiments, the nucleic acid constructs onlycomprise the a construct RNA sequence and may be used independently tobarcode cellular components with origin-specific barcodes without use ofthe fusion proteins and self-reporting export as discussed above. Thesenucleic acid constructs encode a barcode and a cellular RNA captureelement as described above. In certain example embodiments, theconstruct RNA sequence may further comprise a filter sequence. Thefilter sequence is a defined and searchable nucleic acid sequence set ata fixed distance from all barcodes or other unique molecularidentifiers, thus enabling detection of barcodes and unique molecularidentifiers in downstream sequencing data as further described below.The construct RNA sequence may also further comprise an adaptersequence. The adapter sequence defines a nucleic acid sequence that iscomplementary and enables binding of downstream amplification and/orsequencing primers as described further below.

General Construct Elements

In certain example embodiments, all of the constructs disclosed hereinmay further include an inducible promoter to control expression of theconstruct elements. Inducible promoters may include any suitableinducible promoter system. As recognized by one of ordinary skill in theart, the suitability of a particular inducible promoter system isdictated by the cellular system in which the constructs will be used.Accordingly, the biotic or abiotic factors that induce the activity ofsuch promoters must be compatible with the cellular system in which theconstructs of the present invention will be used. For example, a bioticor abiotic factor that negatively impacts cell viability orsignificantly alters gene expression of the cell in the context of thebiological condition being studied would not be a suitable induciblepromoter system. The inducible promoter may be a suitablechemically-regulated promoter or suitable physically-regulated promoter.The chemically-regulated promoter may be a suitable alcohol-regulatedpromoter, tetracycline-regulated promoter, steroid-regulated promoter,or a metal-regulated promoter. The physically-regulated promoters may bea temperature-regulated promoter or a light-regulated promoter. Incertain example embodiments, the inducible promoter is atetracycline-regulated promoter such as pTet-On, pTet-Off, orpTRE-Tight. In certain example embodiments the promoter is adox-inducible promoter. In certain other example embodiments, thepromoter is a cell-specific or tissue-specific promoter. In certainexample embodiments, the construct may comprise both a cell-specific ortissue specific promoter and a second promoter such as dox. See FIG. 5.

In certain example embodiments, all of the constructs disclosed hereinmay further comprise a steric linker sequence. The encoded steric linkersequence may be a random peptide sequence of a particular size. The sizeof the steric linker sequence may control the rate of export, the sizeof the export compartment or both. For example, a larger linker sequenceappended to an export compartment protein may slow the rate at which theexport compartment proteins can self-assemble by creating sterichindrance that slows the rate of assembly. Likewise, a larger linkersequence that must be incorporated into the export compartment mayincrease the size of the export compartment formed. In certain exampleembodiments, the steric linker is approximately 2 to approximately 12amino acids in size. In certain example embodiments, the linker sequenceis located on the N-terminus of the secretion-inducing protein. Incertain other example embodiments, the linker sequence is located on theC-terminus of the secretion-inducing protein.

In certain example embodiments, the constructs disclosed herein mayfurther encode an affinity tag. An affinity tag may include, but is notlimited to, Flag, CBP, GST, HA, HBH, MBP, Myc, polyHis, S-tag, SUMO,TAP, TRX, and V5. Affinity tags may also include engineeredtransmembrane domains in order to increase the likelihood of surfacepresentation. The affinity tags may be then used to purify, for exampleVLPs, formed by the fusion protein using standard affinity purificationtechniques. See FIG. 6. The affinity tag may be encoded by the constructsuch that the affinity tag is located on a N-terminus of thesecretion-inducing protein.

In certain example embodiments, the constructs may further encode anantibiotic resistance gene to facilitate chemical selection of cells orcell populations to which the RNA constructs described herein have beendelivered and expressed. In certain example embodiments, the constructsdisclosed herein may further encode a detectable self-reportingmolecule. In certain example embodiments, the construct may furtherencode a cleavable linker between the detectable self-reporting moleculeand the fusion protein of interest. See FIG. 3. In certain exampleembodiments, the cleavable linker may be a self-cleaving linker such asP2A. In certain example embodiments, the detectable self-reportingmolecule is a fluorescently detectable self-reporting molecule such asRFP, YFP, or GFP. Detection of the self-reporting molecule in a cell orcell population may be used to determine successful delivery andexpression of the constructs disclosed herein.

In certain example embodiments, the construct RNA sequences may furtherencode a nuclear export protein the enables nuclear export of Pol IIIdriven transcript without perturbing cellular localization of otherendogenous RNA transcripts. In certain other example embodiments, thebarcode sequence may be incorporated into the 5′ or 3′ UTR of a Pol IIdriven transcript (e.g. GFP), which is naturally exported to thecytoplasm.

Vectors

In another aspect, the embodiments disclosed herein are directed tovectors for delivering the constructs disclosed herein to cells. Incertain example embodiments the vector is a viral vector. Suitable viralvectors include, but are not limited to, retroviruses, lentiviruses,adenoviruses and AAV. In certain other example embodiments, the vectoris a non-viral vector. Suitable non-viral vectors include, but are notlimited to, cyclodextrin, liposomes, nanoparticles, calcium chloride,dendrimers, and polymers including but not limited to DEAE-dextran andpolyethylenimine. Further non-viral delivery methods includeelectroporation, cell squeezing, sonoporation, optical transfection,protoplast fusion, implalefection, hydrodynamic delivery andmagnetofection. For non-viral vectors, delivery to a microbe may befacilitated by standard transfection technologies such as electricpulsing, electroporation, osmotic shock, and polymeric-based deliverysystems.

Methods of Live Cell Sampling

The constructs and vectors disclosed herein can be used in methods forcontinuous live cell sampling enabling the ability to monitor molecularprofile changes over time. In certain example embodiments, the exportedcellular contents may be barcoded with a cell-specific barcode allowingmultiple samples to be processed in bulk while retaining the ability toidentify the cell or cell population of origin.

In one example embodiment, a method of single cell gene expressionprofiling comprises delivering a nucleic acid construct encoding afusion protein and a construct RNA sequence to a cell or population ofcells. For embodiments utilizing viral vectors, the cell or cells aretransduced with the constructs at a low multiplicity of infection. Incertain example embodiments, the cells may be subsequently subjected tochemical selection to ensure that all cells have a stable single-copy ofthe constructs. For example, the constructs may encode an antibioticresistance gene and chemical selection is carried out by exposure of thecell or cells to a corresponding antibiotic. Alternatively, for thoseembodiments employing a detectable self-reporting molecule, such as GFP,the self-reporting molecule may be used to assess successful. Cellsexpressing the self-reporting molecule may then be selected using knownmethods in the art, such as flow cytometry.

The fusion protein comprises a secretion-inducing domain and a constructRNA capture domain. The construct RNA sequence comprises a retrievalelement and a cellular RNA capture element. The construct RNA sequencemay further comprise a barcode. The barcode comprises a nucleic acidsequence unique to the nucleic acid construct delivered to the cell. Thecellular RNA capture element binds cellular RNA by hybridizing to thecellular RNA. In certain example embodiments the construct RNA sequencehybridizes to mRNA via a poly-U sequence or sequence comprising arepeating (UUG)n motif. In certain example embodiments, thesecretion-inducing domain is an export compartment protein describedherein that self-assembles to form an export compartment. In the processof self-assembling to form the export compartment the construct RNAcapture domain binds the retrieval element on the construct RNA sequenceresulting in the packaging of both the construct RNA sequence and anycellular RNA hybridized to the construct RNA sequence via the constructRNA sequence's cellular retrieval element. The export compartment isthen exported from the cell. For example, the export compartment may bereleased into the cell culture media. The media may then be collectedand the sample isolated. For example, the export compartments may beisolated from the cell culture media by ultracentrifugation, or othermethods that separate components based on size or density. In certainexample embodiments, the fusion protein further comprises an affinitytag as described above, which may be used to isolate and enrich for theexport compartments using standard affinity purification techniquesknown in the art.

The isolated export compartments may then be lysed and the exportedcellular RNAs retrieved. In certain example embodiments, the isolatedVLPs are placed into a hydrogel. The VLPs are then lysed and first andsecond strand synthesis as described above is conducted within thehydrogel. The hydrogel is then dissolved and sequencing librarypreparation conducted as described above. The restrictive diffusionprovided by the hydrogel may be used to prevent potential barcodecross-talk during the RT reaction steps. See FIG. 2

After RNA collection, RNA sequences may be permanently linked to thecellular barcodes by utilizing the barcoded construct RNA sequence as aprimer for reverse transcription thereby incorporating the barcode inthe resulting RNA-DNA duplex. Likewise, in certain example embodiments,the poly-A tail of cellular mRNA may be used to reverse transcribe thebarcode portion of the construct RNA sequence. In certain exampleembodiments a primer designed to bind to the barcode sequence, or aportion thereof, may be used to initiate reverse transcription. SeeFIG. 1. Various example embodiments for incorporation of the barcodesequence into DNA amplicons suitable for sequencing analysis arediscussed below.

Discussion of the following example embodiment is made with reference toFIG. 11. The RNA construct sequence comprises at least, in a 5′ to 3′direction, a retrieval element, a filter, a barcode, and a poly(U) or(UUG)_(n) motif for binding to poly-A tails cellular mRNAs. the RNAconstruct sequence is used to prime first strand cDNA synthesis viareverse transcription of the mRNA template. Template switching may beused to incorporate sequences from a template switching oligonucleotide.For example, a MLV reverse transcriptase—or similar reversetranscriptase—may be used to add non-template nucleotides to thefirst-strand cDNA when it reaches the 5′ end of the mRNA. Templateswitching oligonucleotides designed to bind to these non-templatenucleotides may then be used to facilitate template switching andincorporation of sequences complementary to the template switchingoligonucleotide. In certain example embodiments, the template switchingoligonucleotide may be used to introduce, in a 5′ to 3′ direction, aunique molecular identifier (UMI), a first sequencing primer bindingsite, and an adapter sequence. A UMI is a short nucleotide sequence(e.g. six to eight bp) that uniquely identifies each template switchingoligonucleotide. Next a second cDNA strand is synthesized via reversetranscription and use of a second template switching oligonucleotideresulting in the single stranded cDNA (sscDNA). Double-stranded DNAamplicons suitable for sequencing analysis are then generated byamplification of the sscDNA using the sequencing primer bindingsequences introduced into the sscDNA.

Discussion of the following example embodiment is made with reference toFIG. 12. The construct RNA sequence may comprise, in a 5′ to 3′direction, an adapter sequence a barcode and a poly(U) or (UUG)_(n)motif. Lysis of export compartments may be completed in hydrogels asdescribed above in [0054]. As in the previous embodiment, the constructRNA sequence is used to first prime a reverse transcription reactionthat results in addition of a UMI sequence, sequencing primer bindingsequence and the complement of a RNA polymerase promoter (such as acomplement of a T7 promoter) and the RNA-DNA hybrid show in FIG. 12. Asingle stranded RNA copy is then generated from the RNA-DNA hybrid by invitro transcription with a RNA polymerase and RNA polymerase promoter. Asingle stranded cNDA (sscDNA) is then generated by reverse transcriptionprimed by an adapter primer that binds its complementary sequenceincorporated into the ssRNA. The adapter primer may further comprise asecond UMI and a second sequencing primer binding sequence.Double-stranded DNA amplicons suitable for sequencing analysis are thengenerated by amplification of the dscDNA product using a first andsecond sequencing primer complementary to the first and secondsequencing primer binding sequences.

Discussion of the following example embodiment is made with reference toFIG. 13. The same construct RNA sequence architecture described in[0057] may be use to prime RNA polymerization using T7 RNAP, or similarRNA polymerase, to generate a RNA complement of the cellular mRNA. Areverse transcription reaction is then conducted using a reversetranscription primer, the reverse transcription primer comprising, in a5′ to 3′ direction, a sequencing primer binding sequence and a randomhexamer motif. The resulting RNA comprises the original mRNA sequencewith the random hexamer and first sequencing primer binding sitesequence appended to the 5′ end and the cell barcode and adaptersequence appended to the 3′ end. A single PCR cycle using as secondprimer is conducted to generate a DNA:RNA hybrid, the second primercomprising, in a 5′ to 3′ direction, a second sequencing primer bindingsite, a UMI, and complementary adapter binding sequence. This reactionincorporates the second sequencing primer binding site and UMI into theDNA:RNA hybrid. The DNA:RNA hybrid is then amplified through wholetranscriptome amplification using the first and second sequencingprimers. The resulting dsDNA amplicons may then be prepped forsequencing using standard methods known in the art.

Discussion of the following alternative example embodiments is made withreference to FIG. 14. The construct RNA sequence may comprise, in a 5′to 3′ direction, a barcode a first sequencing primer binding site, apoly(U) or (UUG)_(n) motif. The construct RNA sequence hybridizes to thepoly-A tail of the mRNA via the poly(U) or (UUG)_(n) motif. The 5′ endof the RNA construct sequence is then ligated to the 3′ poly-A tail ofthe mRNA. In certain example embodiments, the mRNA-construct RNA duplexmay be further stabilized prior to ligation by cross-liking the poly-Aand poly(U) sequences, for example using a psoralen. After ligationcross-linking is reversed. The ligated single stranded mRNA product thencomprises, in a 5′ to 3′ direction, the cellular mRNA sequence, barcode,first sequencing primer binding site, and poly(U). The mRNA is reversetranscribed into cDNA as previously described resulting in barcodedcDNA. A second reverse transcription reaction is then primers using aprimer comprising a complementary sequence to the non-templatenucleotides added by the first RT reaction, a UMI, and a secondsequencing primer binding site. The resulting dsDNA product is thenamplified by whole transcriptome amplification using first and secondsequencing primers that hybridize to the first and second sequencingprimer binding sites. The resulting dsDNA amplicons may then be preppedfor sequencing using standard methods known in the art.

Transcripts with the same unique barcode may then be identified asoriginating from the same cell or cell population. Isolated exportcompartments may be collected over multiple time points from the samecells or population of cells. As noted above, the constructs may furtherinclude an inducible promoter to control at what time points theexpression of the export compartment is turned on and off.

In addition, to using sequenced barcode information to identify theorigin of particular transcripts, optical detection of the barcodes mayalso be used to match single-cell gene expression profiles withmicroscopy. Combination with microscopy allows the tissue context of theassayed cells to be derived as well as key measures of cell morphologyand protein levels. For example, optical detection of the barcodes wouldallow relationships between transcriptional changes involving many genesand optically observable phenomena to be tracked in coordinatedtime-lapse measurements at the single-cell level. A set of probes may bederived with each probe cable of specifically hybridizing to a givenoligonucleotide tag in the barcode. Each probe for a givenoligonucleotide sequence may be labeled with a different opticallydetectable label. In one example embodiment, the optically detectablelabel is a fluorophore. In another example embodiment, the opticallydetectable label is a quantum dot. In another example embodiments, theoptically detectable label is an object of a particular size, shape,color, or combination thereof. For each position in the barcode, thecorresponding set of probes for each oligonucleotide tag at thatposition is allowed to hybridize to the cells in situ. The process isrepeated for each position in the barcode. Therefore, the observedpattern of optically detectable barcodes will be dictated by the orderof oligonucleotide sequences in the barcode. Accordingly, the barcodemay be determined by the optical readout obtained with sequentialhybridization of probes.

In certain example embodiments, a set of fluorescently labeled probesspecific to each oligonucleotide tag segment of the barcode may besequentially hybridized to the cells in situ, for example, usingsequential FISH. Each probe is labeled with a different fluorophore.Therefore, the sequence and order of the oligonucleotide tags in thebarcode will dictate the order of colors observed using fluorescencemicroscopy allowing the barcode sequence to be determined optically.

The embodiments are further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—Continuous Monitoring Constructs

Mammalian cells turn over approximately 14% of the transcriptome perhour on average (Yang E, Genome Research 2003), and simulations(described below) show that mRNA can theoretically be exported in VLPsat 100% of the cell's normal synthesis rate. By sampling at 25% of theturnover rate, 3% of the total transcriptome could be sampled per hour,or 500-15,000 transcript molecules per hour. By fine-tuning thetranscriptional and translational dynamics of export compartmentproduction, cellular RNA should be sampled at a specified rate of 0.1%to 3% of the normal synthesis rate. Even with estimated samplepreparation methods that are approximately 50% efficient, detection of250-7500 collected transcript molecules per cell per hour can beachieved. This ‘integration time’ can be varied to resolve the necessarytimescales associated a particular question. A tunable trade-off existsbetween temporal resolution and the degree of perturbation to the cell.

Packing of 28-150 transcripts per VLP inner surface is estimated. Thisestimate is derived from a range in VLP radius of 80-130 nm and an mRNAradius of gyration of 16.8-20.8 nm (mRNA radius of gyration from GopalA, RNA 2012). With these numbers in mind, it is possible to calculatethat the burden of VLP production necessary to collect 15,000 transcriptmolecules per hour corresponds to as little as 0.01% of the cell's totalprotein (total protein per cell count from Siwiak M, PLoS ONE 2013).

To export mRNA in a minimally-biased manner for genome-wide expressionprofiling, a Gag-PABP fusion was constructed and export tested fromHEK293 cells. The construct is safe and replication-deficient, as itcontains neither reverse transcriptase nor integrase. See FIG. 3.Poly(A)-binding protein (PABP), which binds to the poly(A) tail of mRNA,can be used as an mRNA binding domain for synthetic mRNA exportmachinery. The PABP domain will recruit mature transcripts from thecytoplasm, while the Gag domain will allow for export of captured mRNAthrough membrane budding and VLP formation. The overall rate of exportcan be optimized for the desired sampling frequency and cell type bycontrolling the Gag-PABP fusion expression level.

A rate of VLP export of mRNA can be determined by carrying out highlycontrolled VLP collection experiments with an inducible Gag-PABP fusionfrom a known number of cells. RNA from the VLPs can then be extractedand used to prepare RNA-Seq libraries (FIG. 4) with unique molecularidentifiers and a spike-in control (ERCC from Life Technologies). Bycomparing the RNA-seq of bulk cell lysate of self-reporting cells to thelysate of normal cells, the transcriptional defect caused by the VLPexport system can be detected. Similar analysis of the extracted VLPscompared to bulk controls can be used to estimate mRNA export per cellper unit time and any sampling biases (eg against large transcripts).These tests are carried out over a range of different promoter strengthsto find the optimal expression rate, for all cells of interest.

Next, GFP+ self-reporting HEK293 cells are plated in such a way thatthere is 1 cell per well of a 384 well plate on average. To remaincertain that GFP+ cells are self-reporting, GFP and Gag-PABP aredelivered in the same vector. This experiment allows the plate to beimaged to determine the number of GFP+ self-reporting cells, the mediaretrieved to collect VLPs. After collection, VLPs are purified bystandard virus purification protocols. VLP lysis is carried out usingstandard lysis techniques, and lllumina-ready DNA libraries areconstructed using Smart-seq2 (Picelli S, Nature Protocols 2014). Byindexing the media from each well separately through the Smart-seq2protocol, the sequencing reads can be traced to the original wells todetermine the accuracy of VLPs as reporter systems. This can enable GFPexpression as a function of time to be observed, and a correlationbetween GFP reads and cell fluorescence to be determined. The individualcells are collected at the final time point and collected and preparedfor RNA-Seq in the same plate.

Example 2—Barcoded Constructs

Contents from single cells are barcoded by expressing a uniquerandomized RNA sequence with a MS2 hairpin. By hybridizing thesebarcodes to export mRNA, a barcode-mRNA hybrid can be created withreverse transcription after collecting VLPs. To test single-cell mRNAbarcoding and export strategy, a modified version of the collectionmethods described above are used. Gag is fused to a MS2 coat protein,which binds the MS2 RNA hairpin with nanomolar binding affinity. Bytransducing or transfecting cells with a MS2 hairpin containing acell-specific unique random barcode and a 3′ polyU sequence, it ispossible to capture and export mRNA in an unbiased fashion, with eachtranscript stably hybridized to the barcoded MS2 capture probe by thepoly(A):poly(U) interaction. After VLP collection transcript sequencesare permanently linked to the cellular barcodes by utilizing thebarcoded MS2 transcript as a primer for reverse transcription (RT). SuchRNA-primed RT has been previously demonstrated and even shown to resultin higher fidelity than DNA-primed RT (Oude E, JBS 1999). Further,M-MULV RT enzyme has been shown to use both RNA and ssDNA as a template(Verma, B B A 1977), allowing the RNA-DNA hybrids to be convertedcompletely to DNA after a second strand synthesis step with a DNAprimer. See FIG. 1.

The molecular biology steps are tested using in vitro transcribedbarcoded MS2 hairpin RNA and purified total RNA. The (UUG)_(n) motif inthe capture sequence is used to prevent early transcriptionaltermination from pollll promoters, as a stretch of 4 or more uracilbases leads to a 90% transcription termination efficiency (Orioli A, NAR2011). Reverse transcription with a (TTG) DNA primer has been verifiedas efficient as its poly(T) analogue. The in vitro experiment are readout by RT-qPCR of Gapdh-MS2 fusion cDNA. Next, the same assessment isperformed using supernatant from transduced HEK293 cell lysates todemonstrate and optimize endogenous transcript capture by the MS2barcode transcript. Transcript capture and RNA-primed RT from secretedVLPs from bulk HEK293 cultures are tested and complements the RT-qPCRreadout with RNA-Seq of the fusion products (including spike-incontrols) to determine export rates and bias compared with total lysatefrom the same cell population.

Example 3

Single-cell trans-differentiation trajectories can be monitored bydelivering unique RNA barcodes along with the Gag export machinerydescribed here. To do this we can transduce HT1080 fibroblasts withunique RNA barcodes as well as Gag export machinery. Further, can sameHT1080 fibroblasts can be transduced with a MyoD construct to initiatethe trans-differentiation to a myoblast lineage. Bulk populationcontrols and single-cell controls (without export machinery) along thetime course can be used to validate the observed cell-states along eachtrajectory. By collecting supernatant, and building single-cell barcodedlibraries with methods described here, temporal RNA information can betied back to each individual cell of origin. After carrying outdimensionality reduction and other machine learning techniques on theRNAseq data, it is possible to map single-cell trans-differentiationtraj ectories.

Example 4—Nuclear Export of Barcoded Constructions

Self-reporting enables a non-destructive assessment of a cell'stranscriptional state by packaging representative fractions of a cell'stranscriptome into virus-like particles (VLPs), which are subsequentlyexported from the cell into the culture environment. In populationculture of self-reporting cells, genetic encodings may be needed to mapthe RNA exported with VLPs to the cell of origin. Thus, a synthetictransgene was engineered to encode cell state information (e.g. celltype, cell lineage, genetic perturbation, etc.) into an RNAtranscript—termed an RNA barcode—for packaging and export with VLPs. RNAbarcodes are designed to be U6 promoter driven, small RNA transcriptsthat can be stably expressed in cells via viral delivery. Gag viralproteins bind and complex with cytoplasmically expressed RNAs. Thus,nuclear export of the RNA barcode is achieved by including the RevResponse Element (RRE) in the 5′ of the transcript and independentlyco-expressing the HIV-1 Rev viral protein from the same lentiviralvector. Upon expression, Rev protein binds its cognate RRE motif withinthe RNA barcode transcripts to promote Ran-GTP mediated nuclear export.The RNA barcode transcripts also contain MS2 hairpins that can bind theMS2 coat protein (MCP) domain within gag-MCP fusion proteins tospecifically enrich the packaging of RNA barcode transcripts withingag-MCP VLPs. See FIG. 16.

Various modifications and variations of the described methods,pharmaceutical compositions, and kits of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific embodiments, it will be understood that it iscapable of further modifications and that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the art are intended tobe within the scope of the invention. This application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure come within known customarypractice within the art to which the invention pertains and may beapplied to the essential features herein before set forth.

1. A nucleic acid construct comprising a nucleic acid sequence encodinga fusion protein and a construct RNA sequence, the fusion proteincomprising a secretion-inducing domain and a construct RNA sequencecapture domain, the construct RNA sequence comprising a retrievalelement and a cellular RNA capture element, wherein expression of thefusion protein in one or more cells induces export of cellular RNAsbound to the cellular RNA capture element.
 2. The nucleic acid constructof claim 1, wherein the secretion-inducing domain self-assembles uponexpression to form an export compartment.
 3. The nucleic acid constructof claim 1, further encoding one or more of: an inducible promoter tocontrol expression of the nucleic acid sequence encoding the fusionprotein; an affinity tag such that the affinity tag is displayed withthe secretion-inducing domain when expressed; a linker sequence of aparticular size, the size of the linker sequence controlling the rate offormation of an export compartment, a size of the export compartment, orboth; a detectable self-reporting molecule to detect successful deliveryand expression of the nucleic acid constructs; a barcode sequence in theconstruct RNA sequence; and a nuclear export sequence in the constructRNA sequence to facilitate export of construct RNA sequences to thecytoplasm.
 4. The nucleic acid construct of claim 1, wherein thesecretion-inducing domain is a viral capsid protein, and wherein theviral capsid protein is a Gag protein, optionally a lentivirus Gagprotein.
 5. (canceled)
 6. (canceled)
 7. The nucleic acid construct ofclaim 1, wherein the nucleic acid sequence encoding thesecretion-inducing domain is SEQ ID NO:1.
 8. The nucleic acid constructof claim 1, wherein the construct RNA sequence capture domain is MS2coat protein or dCas9.
 9. The nucleic acid construct of claim 1, whereinthe nucleic acid sequence encoding the retrieval domain element of theconstruct RNA sequence is SEQ ID NO:
 10. 10. The nucleic acid constructof claim 1, wherein the retrieval element on the construct RNA sequenceis a dCas9 guide RNA sequence or a MS2 hairpin sequence.
 11. The nucleicacid construct of claim 1, wherein the cellular RNA capture element ofthe construct RNA sequence is a poly-U sequence, and optionally whereinthe poly-U sequence is approximately 15 to approximately 50 nucleotideslong.
 12. (canceled)
 13. The nucleic acid construct of claim 1, whereinthe cellular RNA capture element comprises a (UUG)_(n) motif, andoptionally wherein n is approximately 1 to approximately
 20. 14.(canceled)
 15. The nucleic acid construct of claim 3, wherein thebarcode sequence is a randomized nucleic acid sequence of approximately10 to approximately 40 nucleotides.
 16. The nucleic acid construct ofclaim 1, wherein the secretion-inducing domain self-assembles to form anexport compartment approximately 10 nm to approximately 500 nm indiameter.
 17. A vector comprising the nucleic acid construct of claim 1.18. The vector of claim 17, wherein the vector is a non-viral or viralvector.
 19. (canceled)
 20. A system comprising the nucleic acidconstruct of claim 3 and a nucleic acid construct expressing a nuclearexport protein that facilitates nuclear export of the construct RNAsequence via the nuclear export sequence of the construct RNA sequence.21. The system of claim 20, wherein the nuclear export sequence encodesa viral nuclear export protein, and optionally wherein the viral exportprotein is a Rev viral protein.
 22. (canceled)
 23. A kit comprising thenucleic acid construct of claim
 1. 24. A kit comprising the vector ofclaim
 17. 25. A kit comprising the system of claim
 20. 26. A method forcontinuous monitoring of live cells comprising: delivering into one ormore cells a nucleic acid construct encoding a fusion protein and aconstruct RNA sequence, the fusion protein comprising asecretion-inducing domain and a construct RNA sequence capture domain,and the construct RNA sequence comprising a retrieval element, abarcode, and a cellular RNA capture element; expressing the nucleic acidconstruct in the one or more cells; capturing cellular RNA expressed inthe one or more cells by binding the cellular RNA via the cellular RNAcapture element of the construct RNA sequence; exporting the cellularRNA from the cell by binding of the fusion protein construct RNA captureelement to the retrieval element of the construct RNA such that thecellular RNA is exported from the cell in association with thesecretion-inducing domain, wherein the secretion-inducing domainself-assembles to form an export vesicle; and isolating the exportedvesicles containing captured cellular RNA transcripts at one or moretime points.
 27. The method of claim 26, further comprising: generatinga RNA-DNA duplex by reverse transcribing the captured cell RNAtranscript using the construct RNA sequence as a primer for reversetranscription; generating a DNA-DNA duplex by converting the constructRNA sequence to a corresponding DNA sequence with a second strandsynthesis using a DNA primer such that the barcode sequence is includedin the DNA-DNA duplex; generating a sequencing library from thegenerated DNA-DNA duplexes; sequencing the sequencing library toidentify the captured cell mRNA transcripts wherein the one or morecells from which the cellular RNA transcripts were isolated areidentified from the sequenced barcode.
 28. The method of claim 26,wherein the nucleic acid construct is the nucleic acid construct ofclaim 1, and optionally wherein the nucleic acid construct is deliveredusing a non-viral or a viral vector.
 29. (canceled)
 30. A nucleic acidconstruct encoding a barcode that can be amplified by a cell and used tomark cellular components of the cell according to cell of origin. 31.The nucleic acid construct of claim 30, wherein the nucleic acidconstruct comprises a barcode and a cellular RNA capture element. 32.The nucleic acid construct of claim 31 wherein the cellular RNA captureelement comprises a poly(U) or (UUG)_(n) motif, and optionally whereinthe poly(U) sequence is approximately 15 to approximately 50 nucleotideslong and/or optionally wherein “n” is approximately 1 to approximately20.
 33. (canceled)
 34. The nucleic acid construct of claim 30, whereinthe barcode is a randomized nucleic acid sequence of approximately 10 toapproximately 40 nucleotides.
 35. The nucleic acid construct of claim30, further comprising a searchable filter sequence, wherein the filteris set a fixed distance from the barcode and can be used to identify thebarcode in downstream sequencing reads.
 36. The nucleic acid constructof claim 30, further comprising an adapter sequence, wherein the adapterprovides a complementary binding site for a reverse transcriptionprimer.
 37. The nucleic acid construct of claim 30, further comprising asequencing primer binding site complementary to one or more sequencingprimers.
 38. A vector comprising the nucleic acid construct of claim 30.39. The vector of claim 38, wherein the vector is a non-viral vector ora viral vector.
 40. (canceled)
 41. A kit comprising the nucleic acidconstruct of claim
 30. 42. A kit comprising the vector of claim
 38. 43.A method for labeling molecular components of a cell according to cellof origin, comprising: expressing the nucleic acid construct of claim 30in one or more cells, wherein the barcode is unique to an individualcell or cell lineage; capturing cellular RNA expressed in the one ormore cells by binding the cellular RNA via the cellular RNA captureelement of the expressed construct sequence; and incorporating thebarcode of the expressed nucleic acid construct to the captured cellularRNA to generate barcoded cellular RNA.
 44. The method of claim 43,wherein the barcode is attached to the cellular RNA by use of thenucleic acid construct to prime first strand synthesis of the capturedcellular RNA template.
 45. The method of claim 43, wherein the barcodeis attached to the cellular RNA by ligation of the nucleic acidconstruct to the cellular RNA by RNA-RNA ligation.
 46. The method ofclaim 43, wherein the barcode is attached to the cellular RNA by furtherpriming second strand synthesis.
 47. The method of claim 43, furthercomprising amplifying the barcoded cellular RNA, optionally byRNA-depended RNA synthesis, and wherein the RNA-dependent RNA synthesisis optionally facilitated by T7 RNAP.
 48. (canceled)
 49. (canceled) 50.The method of claim 43, further comprising amplifying the barcodedcellular RNA by PCR.
 51. The method of claim 43, further comprisingamplifying the barcoded cellular RNA by linear DNA amplification.